Stability studies of nucleic acid-binding Fab isolated from combinatorial bacteriophage display libraries

Area of Science:

• Biochemistry
• Molecular Biology
• Immunotechnology

Background:

• Recombinant antibody fragments are crucial tools in molecular biology and diagnostics.
• Bacteriophage display libraries offer a method for generating diverse antibody repertoires.
• Understanding the stability of these fragments is essential for their practical applications.

Purpose of the Study:

• To evaluate the global stability and activity of recombinant DNA-binding antibody fragments.
• To determine if combinatorial assembly and affinity selection favor structurally stable antibody fragments.
• To assess the impact of a C-terminal His tag on fragment stability and binding.

Main Methods:

• Recombinant antibody fragments (Fab) were generated using a bacteriophage combinatorial display library.
• Binding affinities (Kd) to DNA sequences of varying lengths were measured.
• Stability was assessed through thermal denaturation (CD spectroscopy, differential scanning calorimetry), pH, and guanidine-HCI denaturation.
• Irreversible thermal inactivation rates were determined.

Main Results:

• Both tested Fab fragments demonstrated comparable binding affinities (Kd values in the nanomolar range) to different DNA lengths.
• CD spectroscopy and differential scanning calorimetry indicated well-defined tertiary structures, stable below 70°C (Tm 73°C).
• Antibody fragments showed similar rates of thermal inactivation and reversible renaturation from pH and chemical denaturants.

Conclusions:

• Combinatorial antibody fragment production yields stable and active DNA-binding proteins.
• The tested antibody fragments possess robust structural stability, comparable to well-folded proteins.
• These findings support the utility of recombinant antibody fragments in various biotechnological applications.